Nuclear receptors and gene expression in...

Nuclear Receptor Resource White Paper Nuclear receptors and gene expression in liver J.P. Vanden Heuvel, INDIGO Biosciences Inc., State College PA

©2015 Jack Vanden Heuvel, PhD. INDIGO Biosciences, Inc. Reprintpermittedwiththefollowingcitation:JohnP.VandenHeuvel,nrresource.org 1

NuclearreceptorsandgeneexpressioninliverNuclearreceptorsandgeneexpressioninliver.............................................................................11.Overview...................................................................12.ExpressionofNRsintheliver............................23.RoleofNRsinBileAcidMetabolism................24.RoleofNRsinHepaticLipid/GlucoseMetabolism...................................................................24.RoleofNRsinXenobioticMetabolism............45.Specificnuclearreceptorsandtheirroleinhepaticgeneexpression...........................................4a.LiverXReceptors(LXRs)........................................4b.ConstitutiveAndrostaneReceptor(CAR)........5c.PeroxisomeProliferator-ActivatedReceptors(PPARs)...............................................................................6d.PregnaneXReceptor(PXR)...................................7e.FarnesoidXReceptor(FXR)..................................8f.GlucocorticoidReceptor(GR)................................8g.LiverReceptorHomology-1(LRH-1).................9h.RetinoidOrphanReceptorɣ(RORɣ)................10j.ArylhydrocarbonReceptor(AhR)......................11k.Nuclearfactor(erythroid-derived2)-like2(Nrf2)..................................................................................12

6.Citations..................................................................13

1.OverviewNuclear receptors (NR) are a family of ligand-activated transcriptional regulators of several key aspects of hepatic physiology and pathophysiology. In fact, nuclear receptors control a large variety of metabolic processes including hepatic lipid metabolism, drug disposition, bile acid homeostasis, as well as liver regeneration, inflammation, fibrosis, cell differentiation, and tumor formation. Thus, nuclear receptor ligands are being actively explored as novel therapeutic approaches for a broad range of hepatic disorders.

The primary means by which NRs affect such diverse physiological functions is via

alteration of gene transcription. Various NRs sense intracellular molecules, such as bile acids or fatty acids, and regulate intracellular metabolism accordingly. For more information on how NRs regulate gene expression, please consult other White Papers on the NR Resource website (nrresource.org). In this document, we will examine NRs that are involved in gene regulation in the liver, in particular those that are involved in liver disease and drug metabolism.

Figure1.Expressionofnuclearreceptorsinliver(adaptedfromhttps://nursa.org/nursa).NRshighlightedingreenwillbediscussedinmoredetail.



2.ExpressionofNRsintheliverThe primary metabolic cell within the liver is the hepatocyte, although other cells such as Kupffer, Stellate (Ito) and endothelial cells may be involved in responses to diet or xenobiotics. The hepatocyte comprises over 80% of the mass of the liver, and it is the major source of drug and nutrient metabolism. NRs that regulate lipid, glucose, and bile acid homeostasis in the liver are highly expressed in liver (1) (see Figure 1). Of particular note are liver X receptor α (LXRα), constitutive androstane receptor (CAR), peroxisome proliferator activated receptor α (PPARα), pregnane X receptor (PXR), farnesoid X receptor (FXR), glucocorticoid receptor (GR), liver receptor homology-1 (LRH-1), retinoid-related orphan receptor ɣ (RORɣ), hepatic nuclear factor 4α (HNF4α) and the common heterodimer partner Retinoid X receptor α (RXRα). Not shown in Figure 1 are two non-NR transcription factors, the Aryl hydrocarbon Receptor (AhR) and Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which also have roles in liver physiology, in particular xenobiotic metabolism. Following a general discussion of how these receptors are involved in physiological functions in the liver, each receptor and its role in gene regulation will be discussed.

3.RoleofNRsinBileAcidMetabolism NRs play a central role in the regulation of bile acid synthesis, metabolism, and transport (2,3) (Figure 2, panel A). Under cholestatic conditions with high intracellular bile acid load, NRs mediate a coordinated response aimed at protecting hepatocytes from toxic bile acids. Mice lacking the NRs FXR, PXR, and CAR are more vulnerable towards bile acids exposure and cholestatic injury. Genetic variants of FXR may determine susceptibility to gallstones

disease and cholestasis whereas PXR variants have been linked to progression of primary biliary cirrhosis (PBC). Stimulation of the bile acid detoxification machinery with drugs targeting FXR, PXR, and CAR reduces cholestasis and its complications. Such substances are already used in daily clinical practice (e.g., “enzyme inducers” such as rifampicin), whereas others are currently tested in clinical trials and many more are expected to enter clinical trials in the near future. Understanding NR function has therefore not only significantly increased our insights into physiology and pathophysiology of bile acid metabolism but also led to development of NR ligands for the treatment of cholestasis.

4.RoleofNRsinHepaticLipid/GlucoseMetabolismThe liver plays a central role in lipid homeostasis and NRs control several aspects of hepatic lipid and glucose metabolism which may be relevant for the pathogenesis and treatment of metabolic syndrome, hepatic insulin resistance, dyslipidemia, atherosclerosis, and nonalcoholic fatty liver disease (NAFLD) (2,3) (Figure 2, panel A). Several endogenous and exogenous lipids such as cholesterol or fatty acids act as physiological NR ligands and their activation frequently promotes metabolism of respective ligands More specifically, the PPARs and hepatocyte nuclear factor 4α (HNF4α) are activated by various fatty acids, oxidation products of cholesterol such as 24(S)-hydroxycholesterol act as ligands for LXR, and cholesterol metabolites like bile acids act as FXR ligands. Modulation of the activity of these NRs by already available synthetic compounds represents an attractive therapeutic strategy for these metabolic disturbances.



Figure2.RoleofNRsinbileacid,fattyacid,glucoseandxenobioticmetabolism.PanelA.Bileacids(BA)aretakenupbytheNa+/taurocholatecotransporter(NTCP)atthebasolateralmembraneofhepatocytesandareexportedintobilebythecanalicularbilesaltexportpump(BSEP)andthecanalicularconjugateexport pump (MRP2).Thephospholipidexportpump(MDR3)mediatesexcretionofphosphatidylcholine,which formsmixed micelles together with BA and cholesterol in bile and prevents BA-induced bile injury. CYP7A1 is the key enzyme for conversion ofcholesterolintobileacids.Undercholestaticconditions,phaseI(hydroxylationbywayofCYP3A4)andphaseII(conjugationbywayofUGT2B4andSULT2A1)bileaciddetoxificationlimitscholestaticliverinjury.Inaddition,MRP3,MRP4,andtheheteromericorganicsolutetransporterOSTα/βatthebasolateralmembraneofhepatocytesprovideanalternativeexcretionroute forBA intothesystemiccirculation.PanelB.Cholesterol(Chol)metabolites (oxysterols) activate LXR, whereas bile acids (BA) stimulate SHP expression by way of FXR (negative feedback inhibition of BAsynthesis).SHP inhibits itsownexpression ina feedback fashionby targetingLRH-1 (not shown).SREBP-1c (inducedby insulin,LXR/LRH-1,andPPAR-γ) regulatesdenovofattyacid(FA)synthesisfromglucose(Glc).FXR-inducedSHPinhibitsLXR/LRH-1-mediatedtransactivationofSREBP-1cexpression,butalsoindirectlymodulatesSREBP-1cexpression/activitybyalteringcellularcholesterolcontent(notshown).Moreover,SHPtargetsLRH-1-mediated transactivation ofMTP expression, required for triglyceride (TG) assembly with apo B as VLDL triglycerides. Panel C. Nuclearreceptors,aswellasAhRandNrf2areimportanttranscriptionalregulatorsofdrugmetabolismenzymes(DMEs).PhaseIDMEsconsistprimarilyofthecytochromeP450(CYP)superfamilyofmicrosomalenzymesThephaseIImetabolizingorconjugatingenzymes,consistingofmanysuperfamilyof enzymes including sulfotransferases (SULT) and UDP-glucuronosyltransferases (UGT) Phase III transporters, including P-glycoprotein (P-gp),multidrugresistance-associatedprotein(MRP)andorganicaniontransportingpolypeptide2(OATP2).



4.RoleofNRsinXenobioticMetabolismDrug metabolizing enzymes (DMEs) play central roles in the metabolism, elimination and detoxification of xenobiotics and drugs introduced into the human body (4)(Figure 2, panel C). Although most tissues contain a wide array of DMEs including phase I, phase II metabolizing enzymes and phase III transporters, the liver is the predominant site of xenobiotic metabolism. Various NRs as well as the aryl hydrocarbon receptor (AhR) and nuclear factor-erythoroid-related factor 2 (Nrf2) have been shown to be the key regualtors of drug-induced changes in expression of DMEs. For instance, the expression of CYP1 genes can be induced by AhR in response to many polycyclic aromatic hydrocarbon (PAHs). Similarly, the constitutive androstane receptor (CAR) and pregnane X receptor (PXR), both heterodimerize with the retinoid X receptor (RXR), and transcriptionally activate the promoters of CYP2B and CYP3A gene expression by xenobiotics such as phenobarbital-like compounds and dexamethasone and rifampin-type of agents. The peroxisome proliferator activated receptors (PPARs) are activated by lipid lowering, fibrate-type of compounds leading to transcriptional activation of the promoters on CYP4A gene. CYP7A was recognized as the first target gene of the liver X receptor (LXR), in which the elimination of cholesterol depends on CYP7A. Farnesoid X receptor (FXR) was identified as a bile acid receptor, and its activation results in the inhibition of hepatic acid biosynthesis and increased transport of bile acids from intestinal lumen to the liver, and CYP7A is one of its target genes. For the phase II DMEs, phase II gene inducers such as the phenolic compounds butylated hydroxyanisol (BHA), tert-butylhydroquinone (tBHQ) and sulforaphane activate the bZIP transcription

factors Nrf2 and binds to the antioxidant/electrophile response element (ARE) promoter. Phase III transporters, for example, P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs), and organic anion transporting polypeptide 2 (OATP2) play crucial roles in drug absorption, distribution, and excretion. The NRs PXR and CAR, in particular, are involved in the regulation of these transporters. It appears that in general, exposure to phase I, phase II and phase III gene inducers may trigger a cellular “adaptive” response leading to the increase in their gene expression, which ultimately enhance the elimination and clearance of these xenobiotics. Consequently, this homeostatic response of the liver plays a central role in the protection of the body against environmental insults such as those elicited by exposure to xenobiotics. This in turn is also a major means of potential drug-drug interactions. That is, one drug through its interaction with receptors such as PXR, CAR or AhR and consequent induction of DMEs is able to affect the metabolism of a concurrently administered drug or nutrient.

5.Specificnuclearreceptorsandtheirroleinhepaticgeneexpression. As described above, there are several NRs and “xenobiotic-responsive transcription factors” that are highly expressed in the liver. In the following section we will decribe the pathways that affect liver disease (steatosis, cholestasis) and drug-drug interactions.

a.LiverXReceptors(LXRs)The transcriptional factor liver X receptors (LXRs,NR1H2, 3) are involved in cholesterolmetabolism(2). The LXR gene encodes two distinct products,LXRα and LXRβ, each with diverse patterns ofexpression but similar target DNA-binding



elementsand ligands.Theendogenous ligands forLXR are oxysterols (22(R)-hydroxycholesterol,24(S)-hydroxycholesterol, 24(S),25-epoxycholester-ol, 27-hydroxycholesterol) and fatty acids, butthereareseveralsyntheticandantagonistsaswell.Once activated, LXR induces the expression of aclusterof genes that function in lipidmetabolism;these functions are cholesterol absorption, efflux,transport,andexcretion(Figure3).

In addition to itsmetabolic role, LXRs alsomodulate immune and inflammatory responses inmacrophages(2).LXRisakeyregulatorofwhole-body lipidandbileacidmetabolism.LXRregulatesaclusterofgenesthatparticipate inthetransportof excess cholesterol in the form of high-densitylipoprotein (HDL) from peripheral tissue to theliver,aprocesscalledreversecholesteroltransport(RCT). In vivo activation of LXR with a synthetic,high-affinityligandincreasestheHDLlevelandnetcholesterol secretion. LXR positively regulatesseveral enzymes involved in lipoproteinmetabolism including lipoprotein lipase (LPL),humancholesterylestertransportprotein,andthephospholipid transfer protein. LXR also regulatesthecrucialbileacidenzymeCYP7A1.Theenzymaticactivation and conversion of cholesterol to bileacidsisonemechanismforhandlingexcessdietary

cholesterol.

b.ConstitutiveAndrostaneReceptor(CAR)Thehumanconstitutiveandrostanereceptor(CAR,NR1I3) regulates theexpressionof genes involvedinxenobioticmetabolismandtransportintheliver,including CYP2B and 3A4, UGT1 and MDR(5)(Figure4).Studiesfrommousemodelsshowthisnuclear receptor is also involved in bile acid,thyroid hormone and HDL homeostasis. The CARgene uses multiple alternative splicing eventsduring pre-mRNA processing, thereby enhancingthe CAR transcriptome. The 348 amino acid longwild-type human CAR (hCAR1) is encoded by 9exonscomprisedofaDNAbindingdomain(DBD),ahinge region, and a ligand binding domain (LBD).hCAR2 contains an additional four amino acids(VSPT)whilethepredominantvariantexpressedinthe liver, CAR3, contains an additional five aminoacids (APYLT); these insertions are found in theligandbindingdomainofthereceptor.TheratandthemouseCARsequencesaremoresimilartothatofhCAR1andthereislittleevidencetosupporttheexistence of splice variants in these species. ThehCAR2 and hCAR3 transcripts are prominentlyexpressedinhumanliverandprimaryhepatocytes,with combined levels ranging up to ~50%of totalCAR3. Both CAR2 and CAR3 activate reporterscontaining response elements derived from theendogenouspromotersoftheCYP2B6andCYP3A4genes.CAR1isactiveintheabsenceofligandwiththe unique capability to be further regulated byactivators,mainlyviainverseagonism.AnumberofCAR activators, including phenobarbital, do notdirectly bind to the receptor but affect signalingpathwaysthatimpingeontheNR’sactivity.UnlikeCAR1, CAR3 functions as a ligand- dependentreceptor,activatingtranscriptioninthepresenceofthe human CAR ligand 6-(4-chlorophenyl)imi-dazo[2,1-b] thiazole-5-carbaldehyde O-3,4-dichloroben-zyl)oxime (CITCO). CAR2 has a ligandbindingprofilethatisdistinctfrombothCAR1and

Figure3.BasicmechanismofactionofLXRinliver



CAR3,asevidencedbyactivationwithdi-ethylhexylphthalate (DEHP). In addition to splice variantdifferences in activation profiles, speciesdifferences are seen, as shown with the mousespecific CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP). Theexistenceof splicevariants inhumanCARand thespecies differences in activation by xenobioticscertainly makes examining this receptor achallenge.

Activation of the CARmight suppress lipidmetabolismand lowerserumtriglyceride levelsbyreducingthelevelofSREBP-1,amasterregulatoroflipidmetabolism.

c.PeroxisomeProliferator-ActivatedReceptors(PPARs)The peroxisome-proliferator-activated receptors(PPARs;PPARα,PPARβ/δ,andPPARγ,NR1C1-3)areimportant metabolic regulators and have beenutilized as therapeutic targets for treatment ofinsulinresistanceandimpairedfatmetabolism(2).PPARα, PPARβ/δ, and PPARγ exhibit differenttissue distribution and functions and, to someextent,differentligandspecificities.PPARαishighly

expressedintheliver,brownadiposetissue,heart,skeletalmuscle,kidney,andatlowerlevelsinotherorgans. PPARγ is highly expressed in adiposetissues and is present in the colon and lymphoidorgans.PPARβ/δ is expressedubiquitously,but itslevels vary from tissue-to-tissue. Generallyspeaking,theendogenousligandsofthePPARsarefattyacidsortheirmetabolites.WhereasPPARαisregulatedbyawidevarietyof longer-chain lengthfatty acids including saturated, monounsaturatedandpolyunsaturatedfattyacids(PUFA),PPARɣhasmore specificity for PUFAs and their oxidizedmetabolites (6).PPARβ/δ ishasaffinity forPUFAsbutalsofor4-hydroxynonenal,atoxiclipid(7).

Mechanistically, the PPARs formheterodimers with the RXR and activatetranscriptionbybindingtoaspecificDNAelement,termed the peroxisome proliferator responseelement(PPRE),intheregulatoryregionofseveralgenes encoding proteins that are involved in lipidmetabolism and energy balance (see Figure 5). Intheliver,PPARαpromotesfattyacidoxidation.Therole of PPARα in hepatic fatty acidmetabolism isespecially prominent during fasting where PPARαtarget genes such as acyl CoA oxidase (ACO), acylCoA synthase (ACS) and liver-fatty-acid-bindingprotein, are involved inbreakingdown theexcesslipiddeliveredtotheliverduringthisstate.PPARαcanalsoregulateothergenessuchasLPL,whichisinvolved in the degradation of triglycerides, andAPOA1 andAPOCIII,which are both decreased byPPARα agonists. Whereas PPARα controls lipidcatabolism and homeostasis in the liver, PPARγpromotesthestorageoflipidsinadiposetissueandin liver. Intheliver,PPARβ/δisprotectiveagainstliver toxicity induced by environmental chemicals,possibly by downregulating the expression ofproinflammatorygenes.PPARβ/δregulatesglucoseutilization and lipoprotein metabolism bypromotingreversecholesteroltransport.

Figure4.BasicmechanismofactionofCARinliver



d.PregnaneXReceptor(PXR)Predominantly expressed in the liver, Pregnane Xreceptor (PXR,NR1I2) representsoneof themostpromiscuous receptors among the entire NRsuperfamily,andcanbeactivatedbyastructurallydiverse collection of chemicals, including bothxenobioticsandendogenouschemicals.Xenobioticmediated activation of PXR is associatedwith theinduction of many target genes including majorDMEsanddrugtransporters(2,5)(Figure6).TheseincludephaseIenzymessuchasCyps,inparticular

Figure6.BasicmechanismofactionofthePPARsinliver

Figure5.BasicmechanismofactionofPXRinliver



Cyp3A4, phase II enzymes such as UGT1A1 andtransporters including as themultidrug resistanceproteinMDR1P-glycoprotein(Pgp).

In addition to functioning as a xenobioticreceptor and affecting the “adaptive response” tochemical exposure, PXR also influences liverdisease.Forexample,PXR induces lipogenesis inaSREBP-independent manner. Lipid accumulationand marked hepatic steatosis in PXR-transgenicmice are associated with increased expression ofthe fatty acid translocase CD36 and severalaccessory lipogenic enzymes, including SCD-1 andlong-chain free fatty acidelongase.PXRactivationisalsoassociatedwithsuppressionofseveralgenesinvolved in fatty acid β-oxidation, such as PPARαandthiolase.

e.FarnesoidXReceptor(FXR)The main physiological role of the farnesoid Xreceptor(FXR,NR1H4)istoactasabileacidsensorin the enterohepatic tissues (2). FXR activationregulates the expression of various transportproteins and biosynthetic enzymes crucial to thephysiological maintenance of bile acids and lipidandcarbohydratemetabolism(Figure7).Bileacidsbind to and activate this NR with an order ofpotencyofchenodeoxycholicacid>lithocholicacid= deoxycholic acid > cholic acid. In addition toaffecting bile acidmetabolism, FXR reduces bothhepatic lipogenesis and plasma triglyceride andcholesterol levels, induces thegenes implicated inlipoprotein metabolism/clearance, and represseshepatic genes involved in the synthesis oftriglycerides. FXR promotes reverse transport ofcholesterol by increasing hepatic uptake of HDLcholesterol via suppression of hepatic lipaseexpressionandinductionofscavengerreceptorB1,theHDLuptaketransporter in the liver.ActivationoftheFXRalsoincreasesthehepaticexpressionofreceptorssuchasVLDLreceptorandincreasestheexpression of ApoC-II, which coactivateslipoproteinlipase(LPL).Takentogether,thesedata

suggest that FXR activation lowers plasmatriglyceridelevelsviabothrepressingSREBP1-candtriglyceride secretionand increasing the clearanceof triglyceride-rich lipoproteins from the blood. Incarbohydratemetabolism,activationofthehepaticFXRregulatesgluconeogenesis,glycogensynthesis,andinsulinsensitivity.

f.GlucocorticoidReceptor(GR)There is increasing evidence that glucocorticoids(GC) and the glucocorticoid receptor (GR, NR3C1)are importantmediators in liver disease (8). TheclassicalactivationpathwayinvolvesGCbindingtothe GR causing disassociation of the associatedproteincomplexandsubsequenttranslocationintothecellnucleus(Figure8).Followingtranslocation,gene transcription is altered by binding of theGRcomplex to specific DNA sequences known asglucocorticoid response elements (GRE) in thepromoterregionoftargetgenes.Thisalterationingene transcription up-regulates anti-inflammatoryprotein synthesis and reduces expression of pro-inflammatory cytokines. Multiple mechanisms inboth liver and adipose tissue contribute to thedevelopmentof fatty liverdiseaseassociatedwithGRactivity.GCsbind to theGRandaffect lipid inbothadiposeandhepatictissue.Theenzymes11β-HSD&5αR,throughtheirabilitytometabolizeGC

Figure7.BasicmechanismofactionofFXRinliver



can affect GR signaling. In addition, the GC-GRinteraction is regulated by proteins such as Stat5,FKBP52, HES1, MED1, ANGPTL4 and LXRβ. Inadipose tissue, GCs, in the fasting state, mobilizelipidby increasingHormone sensitive Lipase (HSL)and Desnutrin (ATGL - which hydrolysesTriglyceride). In the fedstateGCsare lipogenicbyincreasing expression and activity of DGAT.Expression of Lipases, Acyl Co Carboxylases andFattyAcidSynthases (LPL,ACCandFASN)arealsoincreased.GCsindirectlyaffecthepaticsteatosisbyaltering thereleaseofNEFAsandadipokines fromadipose tissue. GR activation increases de novolipogenesis (DNL) by altering matrix Acyl Co-ADehydrogenase, as well as PEPCK and glucose-6-phosphatedehydrogenase(G6P).GCsincreaseTAGaccumulationbyincreasingexpressionofDGATandreducing TAG Hydrolase (TGH). NEFAs are re-esterifiedintheliverintolipiddroplets.

g.LiverReceptorHomology-1(LRH-1)Liver ReceptorHomolog-1 (LRH-1;NR5A2) is aNRthat plays vital roles in early development and isimportant for bile acid synthesis, cholesterolmetabolismand steroidogenesis. (9). LRH-1bindstoDNAasmonomers tonuclear receptorhalf sitesequences, as shown in Figure 9. LRH-1 was

originally classified as an orphan NR based on itsconstitutive activity and the lack of knownendogenous ligand. This simple view has been inpart revisited with the structures of mouse andhumanLRH-1LBDproteins.Thesestudiesrevealedthat different phospholipid species, includingphosphatidyl glycerol, phosphatidyl ethanolamineand phosphatidyl choline, as well as the secondmessengersphosphatidyl inositols,canbindtothelargeligandbindingpocketofhumanLRH-1(10). A key biological function of LRH-1 is theregulation of cholesterolmetabolism via its effecton bile acid homeostasis (9).. LRH-1 regulatesenterohepatic development and function via theexpressionofkeygenes involved in theregulationofbileacidsynthesis,cholesterolhomeostasisandtransport.LRH-1activatesgenetranscriptionoftherate-limiting enzyme in bile acid biosynthesis,cytochrome P450 family 7A1 or Cholesterol 7 α-hydroxylase (Cyp7A1). LRH-1 positively regulatesexpression of other enzymes and transportersinvolvedinreversetransportofcholesterolandbileacid synthesis pathways. These genes whichcontain the LRH-1 response element (nuclearreceptor half site) in their promoters includecytochrome P450 family 8B1 (Cyp8B1) or Sterol12α hydroxylase, multidrug resistance protein 3(MRP3), cholesteryl ester transfer protein (CETP),scavenger receptor class B type I (SR-BI), mouseapical sodium-dependent bile acid transporter(ASBT) and human Apolipoprotein A1 (ApoA1).Within hepatocytes, cholesterol and cholesterylesters are converted to bile acids by Cyp7A1 andCyp8B1 for secretion out of the liver in bile.Furthermore,MRP3 andASBT are involved in bileacid recycling, indicating that LRH-1 is importantforbileacidhomeostasis.ManyLRH-1genetargetsare involved in the transfer of cholesterol to theliver and subsequent elimination into bile acids,and in bile acid synthesis, highlighting theimportanceofLRH-1incholesterolmetabolism.

Figure8.BasicmechanismofactionofGRinliver



h.RetinoidOrphanReceptorɣ(RORɣ)RORɣconstituteswithRORαandRORβ,theretinoicacid-related orphan receptor (ROR; NR1F1–3)subfamilyofthenuclearreceptors,whichregulatetranscription by binding as monomers to ROR-responsive elements (ROREs) in the regulatoryregion of target genes (11)(Figure 10. Throughalternative promoter usage, the RORγ genegenerates 2 isoforms, RORγ1 and RORγ2 (RORγt),that regulate different physiological functions.RORγtisrestrictedtoseveraldistinctimmunecellsand is essential for thymopoiesis, lymph nodedevelopment,andTh17celldifferentiation.Stearic acid, all-trans retinoic acid (ATRA) and thesynthetic retinoid ALTA 1550 were identified asputativefunctional ligandsandco-crystallizedwithRORβ. Cholesterol and cholesterol sulfate co-crystallized within the ligand binding pocket ofRORα. Crystal structures of RORγ bound to itsagonists 20α-hydroxycholesterol, 22R-hydroxycholesterol and 25-hydroxycholesterolhavebeendetermined.Ursolicacid(UA),anatural

carboxylic acid ubiquitously present in plants is astronginverseagonistofRORγ.

RORγ inverse agonists decrease Th17 celldifferentiation and may provide a noveltherapeuticstrategyinthemanagementofseveralautoimmune diseases. In contrast to RORγt,relatively little is known about the physiologicalfunctions of RORγ1. The expression of RORγ1 ishighly restricted to tissues that have majorfunctions in metabolism and energy homeostasis,including liver and adipose tissue, and in contrastto RORα and RORβ, RORγ is not expressed in thecentral nervous system. By using ubiquitous andliver-specificRORɣ-deficientmiceasmodels,itwasshown that hepatic RORɣ modulates daily insulinsensitivity and glucose tolerance. RORɣ-targetgenes in liver include those linked to glucosehomeostasis(e.g.,G6pase,Pepck,Pklr,Ppard,Gck,Gckr,Glut2,Gys2,Dlat,Pcx,andKlf15).Inaddition,RORγregulatesthecircadianexpressionofInsig2a,Elovl3, Cyp8b1 and other lipid metabolic genes.ThecircadianregulationofRORγexpressionbytheclock machinery together with observations thatRORγ directly regulates the transcription of anumber of lipid metabolic genes, support thehypothesis that RORγ acts downstream of clockproteins and functions as an important linkbetween the circadian clock machinery and itsregulation of certain metabolic genes. RORγ−/−mice display normal cholesterol and triglyceridelevels but slightly reduced blood glucose levelscomparedwiththeirwild-typecounterparts.Thesefindings suggest that RORγ inverse agonists mayhold therapeutic potential for the treatment ofmetabolicsyndromeandassociateddiseases.

Figure9.MechanismofactionofLRH-1inliver



i.HepatocyteNuclearFactor4α(HNF4α)HNF4α(NR2A1),ahighlyconservedmemberofthenuclear receptor superfamily (NR) of ligand-dependenttranscriptionfactors(TFs),isknownasamaster regulator of liver-specific gene expression(12). Basedonthecrystalstructureof theHNF4αligandbindingdomain,endogenousligandsmaybefattyacids;infact,linoleicacid(LA,C18:2ω6)wasfoundboundtoHNF4αinthecrystalizedstructureand subsequent experiments showed it to be aweak agonist. Several CYP450 genes have beenidentified as HNF4α targets with CYP3A4 beingarguably one of the most important (Figure 11).HNF4αisrequiredforbothPXR-andCAR-mediatedtranscriptional activation of CYP3A4. HNF4αactivates the expression of several other Phase Igenes, including CYP2C8, CYP2C9, CYP2C19,CYP7A1andCYP8B1.HNF4αcanbindandactivateFMO1promoteractivityinconcertwithHNF1α.Interms of Phase II genes, HNF4α also plays animportant role in regulating the basal SULT2A1promoter,aswellassynergizingwithPXRandCARTheexpressionoftheUGT1A6andUGT1A9geneswerefoundtobepositivelycorrelatedwithHNF4αand HNF1α expression in human liver. Severaltransporters thatarecritical for theeliminationof

drugs and toxicants (ABCB1, ABCB11, ABCC2,SLCO1B1,SLC22A1),arealsoHNF4αtargetgenes.

j.ArylhydrocarbonReceptor(AhR)TheArylhydrocarbonReceptor (AhR) is amemberof the basic-helix-loop-helix (bHLH)- Per-Arnt-Sim(PAS)genesuperfamilyoftranscriptionfactors(4).AhR is known to recognize a range of chemicalstructures, including non-aromatic and non-halogenated compounds with the prototypicalagonist being 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD). A number of reports have identifiedspecific dietary constituents that are AHR ligands(13). For example, the flavonoids quercetin,apigenin and kaempferol exhibit AHR agonist andantagonist activity in a cell line specific manner.Cruciferousvegetablescontainsignificantamountsof indole glucosinolates that, upon consumption,aredegraded to indole-3-carbinol. This compoundthen undergoes condensation reactions in theacidic environment of the stomach, creatingseveralproductsthatarecapableofactivatingtheAHR,withthecompoundofhighestaffinityfortheAHR being indolo[3,2b]carbazole. The unligandedAhRexistsinthecytosolcomplexedwithadimerofHsp90,whichmaintainstheAhRinaligand-binding

Figure10.MechanismofactionofRORɣinliver

Figure11.MechanismofactionofHNF4αinliver



conformation and prevents nuclear translocationand/or dimerization with Arnt (Figure 12). ThehydrophobicAhRligandsenterthecellbydiffusionand are bound by the Hsp90-associated AhR.Ligand binding causes a conformational changeresulting in a receptor species with an increasedaffinity for DNA. The AhR-Arnt complex toxenobiotic response elements (XRE) or Dioxingresponse elements (DREs) of target genes.Functional XREs, composed of a corepentanucleotide sequence 5ʹ-GCGTG-3ʹ, are foundin the regulatory regions of CYP1A, CYP1B, andUGT1A genes. DNA microarray studies haveestablished that the AhR either directly orindirectly regulates a myriad of genes. Thesestudies have shown that the AhR regulates genesinvolvedinawidevarietyofbiochemicalpathways,includingenergymetabolism, lipidandcholesterolsynthesis, xenobiotic metabolism and varioustransporters. A role of AhR in hepatic lipidmetabolism has been strengthened by the use oftransgenic mice with a constitutively active AhR(CA-AhR) (14). These mice mice exhibitedspontaneous hepatic steatosis, manifested by theaccumulationoftriglyceridesbutnotcholesterolinthe liver. The steatotic effect of AhR wasindependent of SREBP-1c-mediated de novo fattyacidsynthesis. Instead,the lipogeniceffectofAhRlikely resulted from its activation of CD36 geneexpressionandtheconsequentincreaseofhepaticFFAuptake.InadditiontoCD36,theexpressionofFATP1andFATP2,whichcanalsofacilitatehepaticFFA uptake, was increased in CA-AhR transgenicmice.

k.Nuclearfactor(erythroid-derived2)-like2(Nrf2)Nuclear erythroid 2-related factor 2 (Nrf2) is atranscriptionfactorthatbelongstotheCap-n-collar

basicleucinezipperfamily(15).Nrf2isconsideredthemainmediatorof cellular adaptation to redoxstress. In its inactive state, Nrf2 is located in thecytoplasmwhereitinteractswiththeactinbindingprotein,Kelch-likeECHassociatingprotein1,andisrapidly degraded by the ubiquitin-proteasomepathway.KEAP1hasmorethan20freesulfhydryl(-SH)groupsinitsconstituentcysteineresiduesthatact as stress sensors. Various oxidative orelectrophilic cellular stresses, including ROS andreactive nitrogen species (RNS), modify KEAP1cysteine residues, causing their dissociation andsubsequent translocation of Nrf2 to the nucleus(Figure 13). In the nucleus, Nrf2 binds to AREsequencesandfunctions inpartnershipwithothernuclear proteins as a strong transcriptionalactivator of ARE-responsive genes. ARE-mediatedantioxidant proteins and enzymes, such as hemeoxygenase-1 (HO-1), NAD(P)H: quinoneoxidoreductase 1 (NQO1), glutathione-S-transferases(GST),groupCstreptococcus(GCS)areinvolved in the detoxification of increasedelectrophilesandradicals.

Figure12.MechanismofactionofAhRinliver



6.Citations1.Bookout,A.L.,Jeong,Y.,Downes,M.,Yu,R.T.,Evans,R. M., and Mangelsdorf, D. J. (2006) Anatomicalprofiling of nuclear receptor expression reveals ahierarchicaltranscriptionalnetwork,Cell126,789-799.2.López-Velázquez,J.A.,Carrillo-Córdova,L.D.,Chávez-Tapia,N.C.,Uribe,M.,andMéndez-Sánchez,N.(2012)Nuclear receptors in nonalcoholic Fatty liver disease, JLipids2012,139875.3. Wagner, M., Zollner, G., and Trauner, M. (2011)Nuclearreceptorsinliverdisease,Hepatology53,1023-1034.4. Xu, C., Li, C. Y., and Kong, A.N. (2005) Induction ofphase I, II and III drug metabolism/transport byxenobiotics,ArchPharmRes28,249-268.5.Omiecinski,C.J.,VandenHeuvel,J.P.,Perdew,G.H.,and Peters, J. M. (2011) Xenobiotic metabolism,disposition, and regulation by receptors: frombiochemical phenomenon to predictors of majortoxicities,ToxicolSci120Suppl1,S49-S75.6.Gillies,P.J.,Bhatia,S.K.,Belcher,L.A.,Hannon,D.B.,Thompson, J. T., and Vanden Heuvel, J. P. (2012)Regulationofinflammatoryandlipidmetabolismgenesby eicosapentaenoic acid-rich oil, J Lipid Res 53, 1679-1689.7.Coleman,J.D.,Prabhu,K.S.,Thompson,J.T.,Reddy,P. S., Peters, J. M., Peterson, B. R., Reddy, C. C., andVanden Heuvel, J. P. (2007) The oxidative stress

mediator4-hydroxynonenalisanintracellularagonistofthe nuclear receptor peroxisome proliferator-activatedreceptor-beta/delta (PPARbeta/delta), Free Radic BiolMed42,1155-1164.8.Woods,C.P.,Hazlehurst,J.M.,andTomlinson,J.W.(2015) Glucocorticoids and non-alcoholic fatty liverdisease,JSteroidBiochemMolBiol154,94-103.9. Lazarus, K. A., Wijayakumara, D., Chand, A. L.,Simpson, E. R., and Clyne, C. D. (2012) Therapeuticpotential of Liver Receptor Homolog-1 modulators, JSteroidBiochemMolBiol130,138-146.10.Stein,S.,andSchoonjans,K.(2015)Molecularbasisfor the regulation of the nuclear receptor LRH-1, CurrOpinCellBiol33,26-34.11.Takeda,Y.,Kang,H. S., Freudenberg, J.,DeGraff, L.M., Jothi, R., and Jetten, A. M. (2014) Retinoic acid-relatedorphanreceptorγ(RORγ):anovelparticipantinthe diurnal regulation of hepatic gluconeogenesis andinsulinsensitivity,PLoSGenet10,e1004331.12. Hwang-Verslues, W. W., and Sladek, F. M. (2010)HNF4α--role in drug metabolism and potential drugtarget?,CurrOpinPharmacol10,698-705.13. Murray, I. A., Patterson, A. D., and Perdew, G. H.(2014) Aryl hydrocarbon receptor ligands in cancer:friendandfoe,NatRevCancer14,801-814.14. He, J., Lee, J. H., Febbraio,M., and Xie,W. (2011)The emerging roles of fatty acid translocase/CD36 andthearylhydrocarbonreceptorinfattyliverdisease,ExpBiolMed(Maywood)236,1116-1121.15.Tang,W.,Jiang,Y.F.,Ponnusamy,M.,andDiallo,M.(2014) Role of Nrf2 in chronic liver disease, World JGastroenterol20,13079-13087.

Figure13.MechanismofactionofNrf2inliver

Nuclear receptors and gene expression in...

Documents

Transcript of Nuclear receptors and gene expression in...